ABSTRACT Rhythm is a critical feature of locomotion and is generated by interneurons in the spinal cord. The intrinsic and local mechanisms employed by lumbar spinal rhythmogenic interneurons and how they are recruited by supraspinal locomotor centers represent major gaps in our understanding of rhythmogenesis and locomotor circuitry. This information is crucial in the pursuit of therapeutic targets to treat the leading causes of paralysis including spinal cord injury, traumatic brain injury, and Parkinson’s Disease. Spinal interneurons expressing the transcription factor Shox2 include a group of putatively rhythmogenic interneurons in the mouse. Shox2 interneurons in the adult lumbar spinal slice possess rhythmogenic ionic currents, including persistent inward currents, and make functional excitatory connections to other Shox2 interneurons. We have found that a subset of Shox2 interneurons in the adult lumbar spinal slice displays spontaneous rhythmic membrane potential oscillations. Intrinsic and local network properties are essential for the initiation and maintenance of rhythmic oscillations in other models of neuronal bursting and Shox2 interneurons in the spinal slice allow for the study of the specific mechanisms involved in the adult mammalian locomotor circuitry. This proposal explores how oscillations in Shox2 interneurons are generated and recruited using an innovative approach that combines whole cell patch clamp electrophysiology, transsynaptic tract tracing, and optogenetics. With this combinatorial approach, we will test the overarching hypothesis that Shox2 interneurons in the lumbar spinal cord of adult mice display rhythmic firing that is critically mediated by persistent sodium current and local excitatory synaptic connections and recruited by monosynaptic excitatory input from the lateral paragigantocellular nucleus in the brainstem. In whole-cell patch clamp experiments, we will identify the voltage sensitive current(s) and underlying voltage-gated ion channels critically involved in rhythmic oscillations in individual Shox2 interneurons. Additionally, we will pharmacologically test the contributions of the local synaptic connections to oscillatory properties in Shox2 INs in the lumbar spinal slice. Lastly, the supraspinal nuclei which monosynaptically project to lumbar Shox2 interneurons will be identified by monosynaptic restricted transsynaptic tracing from Shox2 interneurons in the adult mouse. These anatomical projections will be functionally evaluated optogenetically in the adult lumbar spinal slice. Together, this represents essential first steps in identifying and evaluating mechanisms of rhythm generation in and recruitment of Shox2 interneurons which may serve as therapeutic targets for the treatment of paralysis in which spinal locomotor circuits are left intact, but dormant.